Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation.

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.

ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism.

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3-/-) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3-/- cells; rather ANGPTL3-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3-/-;LDLR-/- cells rescued the deficient LDL clearance of LDLR-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

Quercetin inhibits NF-kB and JAK/STAT signaling via modulating TLR in thymocytes and splenocytes during MSG-induced immunotoxicity: an in vitro approach.

BACKGROUND: The most widely used food additive monosodium glutamate (MSG) has been linked to immunopathology. Conversely, quercetin (Q), a naturally occurring flavonoid has been demonstrated to have immunomodulatory functions. Therefore, the purpose of the study is to determine if quercetin can mitigate the deleterious effects of MSG on immune cells, and the possible involvement of TLR, if any.  METHODS AND RESULTS: This study was conducted on Q, to determine how it affects the inflammatory response triggered by MSG in primary cultured thymocytes and splenocytes from rats (n = 5). Q shielded cells by augmenting cell survival and decreasing lactate dehydrogenase leakage during MSG treatment. It decreased IL-1ß, IL-6, IL-8, and TNF-α expression and release by hindering NF-kB activation and by inhibiting the JAK/STAT pathway. Moreover, Q prevented NLRP3 activation, lowered IL-1ß production, and promoted an anti-inflammatory response by increasing IL-10 production. Q reduced MSG-induced cellular stress and inflammation by acting as an agonist for PPAR-γ and LXRα, preventing NF-kB activation, and lowering MMP-9 production via increasing TIMP-1. Additionally, Q neutralized free radicals, elevated intracellular antioxidants, and impeded RIPK3, which is involved in inflammation induced by oxidative stress, TNF-α, and TLR agonists in MSG-treated cells. Furthermore, it also modulated TYK2 and the JAK/STAT pathway, which exhibited an anti-inflammatory effect. CONCLUSIONS: MSG exposure is associated with immune cell dysfunction, inflammation, and oxidative stress, and Q modulates TLR to inhibit NF-kB and JAK/STAT pathways, providing therapeutic potential. Further research is warranted to understand Q's downstream effects and explore its potential clinical applications in inflammation.

ABDpred: Prediction of active antimicrobial compounds using supervised machine learning techniques.

BACKGROUND OBJECTIVES: Discovery of new antibiotics is the need of the hour to treat infectious diseases. An ever-increasing repertoire of multidrug-resistant pathogens poses an imminent threat to human lives across the globe. However, the low success rate of the existing approaches and technologies for antibiotic discovery remains a major bottleneck. In silico methods like machine learning (ML) deem more promising to meet the above challenges compared with the conventional experimental approaches. The goal of this study was to create ML models that may be used to successfully predict new antimicrobial compounds. METHODS: In this article, we employed eight different ML algorithms namely, extreme gradient boosting, random forest, gradient boosting classifier, deep neural network, support vector machine, multilayer perceptron, decision tree, and logistic regression. These models were trained using a dataset comprising 312 antibiotic drugs and a negative set of 936 non-antibiotic drugs in a five-fold cross validation approach. RESULTS: The top four ML classifiers (extreme gradient boosting, random forest, gradient boosting classifier and deep neural network) were able to achieve an accuracy of 80 per cent and above during the evaluation of testing and blind datasets. INTERPRETATION CONCLUSIONS: We aggregated the top performing four models through a soft-voting technique to develop an ensemble-based ML method and incorporated it into a freely accessible online prediction server named ABDpred ( http://clinicalmedicinessd.com.in/abdpred/ ).

Fate of the unoperated ascending thoracic aortic aneurysm: three-decade experience from the Aortic Institute at Yale University.

AIMS: This study aims to outline the 'true' natural history of ascending thoracic aortic aneurysm (ATAA) based on a cohort of patients not undergoing surgical intervention. METHODS AND RESULTS: The outcomes, risk factors, and growth rates of 964 unoperated ATAA patients were investigated, over a median follow-up of 7.9 (maximum of 34) years. The primary endpoint was adverse aortic events (AAE), including dissection, rupture, and aortic death. At aortic sizes of 3.5-3.9, 4.0-4.4, 4.5-4.9, 5.0-5.4, 5.5-5.9, and ≥6.0 cm, the average yearly risk of AAE was 0.2%, 0.2%, 0.3%, 1.4%, 2.0%, and 3.5%, respectively (P < 0.001), and the 10-year survival free from AAE was 97.8%, 98.2%, 97.3%, 84.6%, 80.4%, and 70.9%, respectively (P < 0.001). The risk of AAE was relatively flat until 5 cm of aortic size, at which it began to increase rapidly (P for non-linearity <0.001). The mean annual growth rate was estimated to be 0.10 ± 0.01 cm/year. Ascending thoracic aortic aneurysms grew in a very slow manner, and aortic growth over 0.2 cm/year was rarely seen. Multivariable Cox regression identified aortic size [hazard ratio (HR): 1.78, 95% confidence interval (CI): 1.50-2.11, P < 0.001] and age (HR: 1.02, 95% CI: 1.00-1.05, P = 0.015) as significant independent risk factors for AAE. Interestingly, hyperlipidemia (HR: 0.46, 95% CI: 0.23-0.91, P = 0.025) was found to be a significant protective factor for AAE in univariable Cox regression. CONCLUSION: An aortic size of 5 cm, rather than 5.5 cm, may be a more appropriate intervention criterion for prophylactic ATAA repair. Aortic growth may not be an applicable indicator for intervention.

"Big Data" Analyses Underlie Clinical Discoveries at the Aortic Institute.

This issue of the Yale Journal of Biology and Medicine (YJBM) focuses on Big Data and precision analytics in medical research. At the Aortic Institute at Yale New Haven Hospital, the vast majority of our investigations have emanated from our large, prospective clinical database of patients with thoracic aortic aneurysm (TAA), supplemented by ultra-large genetic sequencing files. Among the fundamental clinical and scientific discoveries enabled by application of advanced statistical and artificial intelligence techniques on these clinical and genetic databases are the following: From analysis of Traditional "Big Data" (Large data sets). 1. Ascending aortic aneurysms should be resected at 5 cm to prevent dissection and rupture. 2. Indexing aortic size to height improves aortic risk prognostication. 3. Aortic root dilatation is more malignant than mid-ascending aortic dilatation. 4. Ascending aortic aneurysm patients with bicuspid aortic valves do not carry the poorer prognosis previously postulated. 5. The descending and thoracoabdominal aorta are capable of rupture without dissection. 6. Female patients with TAA do more poorly than male patients. 7. Ascending aortic length is even better than aortic diameter at predicting dissection. 8. A "silver lining" of TAA disease is the profound, lifelong protection from atherosclerosis. From Modern "Big Data" Machine Learning/Artificial Intelligence analysis: 1. Machine learning models for TAA: outperforming traditional anatomic criteria. 2. Genetic testing for TAA and dissection and discovery of novel causative genes. 3. Phenotypic genetic characterization by Artificial Intelligence. 4. Panel of RNAs "detects" TAA. Such findings, based on (a) long-standing application of advanced conventional statistical analysis to large clinical data sets, and (b) recent application of advanced machine learning/artificial intelligence to large genetic data sets at the Yale Aortic Institute have advanced the diagnosis and medical and surgical treatment of TAA.

Dichlorophene activates aryl hydrocarbon receptor (AhR) and indoleamine 2, 3-dioxygenase 1 (IDO1) to mediate splenotoxicity in rat.

Dichlorophene (DCP) is a halogenated phenolic compound, widely used as fungicide, bactericide and antiprotozoan and also exhibit therapeutic application in several pathological conditions. Taking account of broad use of DCP, its possible effect on spleen (an important immune organ) was investigated in this study. Male albino rats were treated with graded doses of DCP (10%, 20% and 30% of LD50) and spleen and blood were obtained at 24, 48 and 72 hours post treatment. Oxidative stress parameters, proinflammatory cytokines and protein expression of aryl hydrocarbon receptor (AhR), indoleamine-2, 3-Dioxygenase 1 (IDO1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were measured along with histopathological evaluation of spleen. In the present study, DCP perturbs redox status of splenocytes of rats as evidenced by excess ROS generation, lipid peroxidation and nitric oxide production simultaneously with reduction of antioxidant level [glutathione (GSH)] and inhibition of antioxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)]. Two important proinflammatory cytokines, IL-6 and TNF-α were found to be elevated upon DCP treatment. Moreover, DCP also caused activation of AhR and IDO1 with simultaneous down regulation of Nrf2. All these effects of DCP were found to be dose and duration dependent. DCP also affects the spleen micro-architecture in the present study and these alterations were more prominent in high dose group at 72 hours post treatment. Taken together, all these results suggested that DCP induces oxidative stress and also increases proinflammatory cytokine levels to mount its toxic effect on spleen.

Whole Body Ip6k1 Deletion Protects Mice from Age-Induced Weight Gain, Insulin Resistance and Metabolic Dysfunction.

(1) Background: We previously demonstrated that disruption of IP6K1 improves metabolism, protecting mice from high-fat diet-induced obesity, insulin resistance, and non-alcoholic fatty liver disease and steatohepatitis. Age-induced metabolic dysfunction is a major risk factor for metabolic diseases. The involvement of IP6K1 in this process is unknown. (2) Methods: Here, we compared body and fat mass, insulin sensitivity, energy expenditure and serum-, adipose tissue- and liver-metabolic parameters of chow-fed, aged, wild type (aWT) and whole body Ip6k1 knockout (aKO) mice. (3) Results: IP6K1 was upregulated in the adipose tissue and liver of aWT mice compared to young WT mice. Moreover, Ip6k1 deletion blocked age-induced increase in body- and fat-weight and insulin resistance in mice. aKO mice oxidized carbohydrates more efficiently. The knockouts displayed reduced levels of serum insulin, triglycerides, and non-esterified fatty acids. Ip6k1 deletion partly protected age-induced decline of the thermogenic uncoupling protein UCP1 in inguinal white adipose tissue. Targets inhibited by IP6K1 activity such as the insulin sensitivity- and energy expenditure-inducing protein kinases, protein kinase B (PKB/Akt) and AMP-activated protein kinase (AMPK), were activated in the adipose tissue and liver of aKO mice. (4) Conclusions: Ip6k1 deletion maintains healthy metabolism in aging and thus, targeting this kinase may delay the development of age-induced metabolic dysfunction.

Interstage mortality in two-stage elephant trunk surgery.

PURPOSE: Diffuse mega-aorta is challenging. Prior studies have raised concerns regarding the safety of the open two-stage elephant trunk (ET) approach for extensive thoracic aortic aneurysm (TAA), specifically in regard to interstage mortality. This study evaluates the safety of the two-stage ET approach for management of extensive TAA. METHODS: Between 2003 and 2018, 152 patients underwent a Stage I ET procedure by a single surgeon (mean age 64.5 ± 14.8). Second stage ET procedure was planned in 60 patients (39.4%) and to-date has been performed in 54 patients (90%). (in the remaining patients, the ET was prophylactic for the long-term, with no plan for near-term utilization). RESULTS: In-hospital mortality after the Stage I procedure was 3.3% (5/152). In patients planned for Stage II, the median interstage interval was 5 weeks (range: 0-14). Of the remaining six patients with planned, but uncompleted Stage II procedures, five patients expired from various causes in the interval period (interstage mortality of 8.3%). There were no cases of aortic rupture in the interstage interval. Stage II was completed in 58 patients (including four unplanned) with a 30-day mortality of 10.3% (6/58). Seven patients developed strokes after Stage II (12%), and three patients (5.1%) developed paraplegia. CONCLUSIONS: The overall mortality, including Stage I, interstage interval, and Stage II was 18.6%. This substantial cumulative mortality for the open two-staged ET approach for the treatment of extensive TAA appears commensurate with the severity of the widespread aortic disease in this patient group. Fear of interstage rupture should not preclude the aggressive Two-Stage approach to the management of extensive TAA.

A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

Targeting the Inositol Pyrophosphate Biosynthetic Enzymes in Metabolic Diseases.

In mammals, a family of three inositol hexakisphosphate kinases (IP6Ks) synthesizes the inositol pyrophosphate 5-IP7 from IP6. Genetic deletion of Ip6k1 protects mice from high fat diet induced obesity, insulin resistance and fatty liver. IP6K1 generated 5-IP7 promotes insulin secretion from pancreatic ß-cells, whereas it reduces insulin signaling in metabolic tissues by inhibiting the protein kinase Akt. Thus, IP6K1 promotes high fat diet induced hyperinsulinemia and insulin resistance in mice while its deletion has the opposite effects. IP6K1 also promotes fat accumulation in the adipose tissue by inhibiting the protein kinase AMPK mediated energy expenditure. Genetic deletion of Ip6k3 protects mice from age induced fat accumulation and insulin resistance. Accordingly, the pan IP6K inhibitor TNP [N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates obesity, insulin resistance and fatty liver in diet induced obese mice by improving Akt and AMPK mediated insulin sensitivity and energy expenditure. TNP also protects mice from bone loss, myocardial infarction and ischemia reperfusion injury. Thus, the IP6K pathway is a potential target in obesity and other metabolic diseases. Here, we summarize the studies that established IP6Ks as a potential target in metabolic diseases. Further studies will reveal whether inhibition of this pathway has similar pleiotropic benefits on metabolic health of humans.

Functionally significant metabolic differences between B and T lymphocyte lineages.

Activation of B and T lymphocytes leads to major remodelling of the metabolic landscape of the cells enabling their post-activation functions. However, naive B and T lymphocytes also show metabolic differences, and the genesis, nature and functional significance of these differences are not yet well understood. Here we show that resting B-cells appeared to have lower energy demands than resting T-cells as they consumed lower levels of glucose and fatty acids and produced less ATP. Resting B-cells are more dependent on OXPHOS, while T-cells show more dependence on aerobic glycolysis. However, despite an apparently higher energy demand, T lineage cells showed lower rates of protein synthesis than equivalent B lineage stages. These metabolic differences between the two lineages were established early during lineage differentiation, and were functionally significant. Higher levels of protein synthesis in B-cells were associated with increased synthesis of MHC class II molecules and other proteins associated with antigen internalization, transport and presentation. The combination of higher energy demand and lower protein synthesis in T-cells was consistent with their higher ATP-dependent motility. Our data provide an integrated perspective of the metabolic differences and their functional implications between the B and T lymphocyte lineages.

Vector-free in vivo trans-determination of adult hepatic stem cells to insulin-producing cells.

A reduction in the number of functional ß-cells is the central pathological event in diabetes. Liver and ventral pancreas differentiates simultaneously in the same general domain of cells within embryonic endoderm. In addition, the precursor cell population being bi-potential may be targeted for either pancreas or liver development. Hepatic stem cells were redirected in vivo to functional insulin producing cells in a acetylaminofluorene-partial hepatectomy (AAF/PH) adult male rat model with/without GLP-1 treatment. In routine H&E histology and immunohistochemistry, stem cells resembled ß cells in GLP-1 injected rats. Immunoblots revealed involvement of adenylate cyclase, TLR4 and PDX1 in insulin synthesis. Expression of genes (GLP-1R, MAFA, PDX1, INS1 and INS2) augmented in the GLP-1 treated regenerated liver. Results strongly indicated the key role of GLP-1 in the induction of insulin secretion in trans-determined reprogrammed cell in vivo. The present method being vector free poses no risk of vector spillover in the host and holds promise.

Excess iodine-induced lymphocytic impairment in adult rats.

Altered lymphocytic activity and its subset ratio found responsible for initiating abnormal autoimmune responses in men and animals after excess iodine exposure. Study objective is to reveal excess iodine-induced impairment of peripheral blood lymphocytes (PBL), its functional status, antioxidant balance, DNA damage, proliferation assay, and serum cytokine levels (IL6 and TNF α)in adult male rats to understand the onset of autoimmune alterations if any indirectly that is unexplored. Experimental animals were grouped depending on doses of iodine(KI) treatment with moderately excess-7 mg/kg bw (100EI) and excessively excess-35 mg/kg bw (500EI)for 30 days to analyze IL6 and TNF α, hematological indices, oxidative stress, lymphocytic DNA damage, and proliferation status. Significant impairment in superoxide dismutase, catalase, GPx activities including elevated NO, LPO in lymphocytes of treated group, with increased IL6 and TNF α level, lymphocyte proliferation and DNA damage depending on doses of iodine. Therefore, excess iodine consumption leads to lymphocytic impairment that may be the potential cause of autoimmune thyroid diseases in long run. Highlights Excess iodine triggers the oxidative stress in lymphocytes. Excess iodine promotes the activity of pro-inflammatory cytokines. Excess iodine causes impairment of functional status of lymphocytes leading to immune-cytotoxicity. Excess iodine exacerbates the autoimmunity.

Attenuation of macrophage accumulation and polarisation in obese diabetic mice by a small molecule significantly improved insulin sensitivity.

Accumulation and polarization of anti-inflammatory M2 to proinflammatory M1 macrophage in the adipose tissue of obese diabetic mice is an important pathogenic signature. It worsens lipid induced inflammation and insulin resistance. Here we demonstrate that a small molecule, a peroxyvanadate compound i.e. DmpzH [VO(O2)2 (dmpz)] or dmp, could robustly decrease macrophage infiltration, accumulation and their polarization in high fat diet (HFD) induced obese diabetic mice. In searching the underlying mechanism it was revealed that SIRT1 level was strikingly low in the inflamed adipose tissue of HFD mice as compared to mice fed with standard diet (SD). Administration of dmp markedly increased SIRT1 level by inducing its gene expression with a consequent decrease in macrophage population. Elevation of SIRT1 coincided with the decrease of MCP1, Fetuin-A (FetA) and IFNγ. Since MCP1 and FetA drive macrophage to inflamed adipose tissue and IFNγ promotes M2 to M1 transformation, both recruitment and M1 induced inflammation were found to be significantly repressed by dmp. In addressing the question about how dmp induced excess SIRT1 could reduce MCP1, FetA and IFNγ levels, we found that it was due to the inactivation of NFκB because of its deacetylation by SIRT1. Since NFκB is the transcriptional regulator of these molecules, their expressions were significantly suppressed and that caused sharp decline in macrophage recruitment and their polarity to M1. This effected a marked fall in proinflammatory cytokine level which significantly improved insulin sensitivity. dmp is likely to be the first molecule that rescues inflammatory burden contributed by macrophage in obese diabetic mice adipose tissue which causes significant increase in insulin sensitivity therefore it may be a meaningful choice to treat type 2 diabetes.

Impact of edaphic factors and nutrient management on the hepatoprotective efficiency of Carlinoside purified from pigeon pea leaves: An evaluation of UGT1A1 activity in hepatitis induced organelles.

Carlinoside is a unique compound well-known for its excellent curative potential in hepatitis. There is a substantial research gap regarding the medicinal use of carlinoside, as its concentrations are greatly variable (depending on locality). We cultivated Cajanus cajan using vermicompost as a major organic amendment at two locations (Sonitpur and Birbhum) with different soil types, but identical climate conditions. Sonitpur soils were richer in soil organic C (SOC), enzyme activation, and N/P content than Birbhum. However, vermi-treatment improved many soil properties (bulk density, water retention, pH, N/P/K, and enzyme activity) to narrow the locational gap in soil quality by 15-28%. We also recorded a many-fold increment in SOC storage capacities in both locations, which was significantly correlated with carlinoside, total phenol, and flavonoid contents in Cajanus leaves. This significantly up-regulated the carlinoside induced expression of the bilirubin-solubilizing UGT1A1enzyme in HepG2 cell and rat liver. Leaf extracts of vermicompost-aided plants could cure hepatitis in affected rat livers and in the HepG2 cell line. Accordingly, vermi-treatment is an effective route for the growth of Cajanus as a cash crop for biomedical applications and can produce a concurrent improvement in soil quality.

Targeting mitochondria with folic acid and vitamin B12 ameliorates nicotine mediated islet cell dysfunction.

Nicotine, one of the well-known highly toxic components of cigarette smoke, causes a number of adverse health effects and diseases. Our previous study has shown that nicotine induces reactive oxygen species (ROS) in islet cell and disrupts islet cell mitochondrial membrane potential (ΔΨm). However, supplementation with folic acid and vitamin B12 were found effective against nicotine induced changes in pancreatic islet cells. But the toxicological effects and underlying mechanisms of nicotine-induced mitochondrial dysfunction is still unknown. In this study, nicotine exposure decreases mitochondrial enzymes (pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, aconitase, malate dehydrogenase) activities by increasing cytosolic Ca2+ level which may contribute to increased mitochondrial ROS production by raising its flow to mitochondria. This in turn produces malondialdehyde and nitric oxide (NO) with a concomitant decrease in the activities of antioxidative enzymes and glutathione levels leading to loss of ΔΨm. Simultaneously, nicotine induces pancreatic islet cell apoptosis by modulating ΔΨm via increased cytosolic Ca2+ level, altered Bcl-2, Bax, cytochrome c, caspase-9, PARP expressions which were prevented by the supplementation of folic acid and vitamin B12 . In conclusion, nicotine alters islet cell mitochondrial redox status, apoptotic machinery, and enzymes to cause disruption in the ΔΨm and supplementation of folic acid and vitamin B12 possibly blunted all these mitochondrial alterations. Therefore, this study may help to determine the pathophysiology of nicotine-mediated islet cell mitochondrial dysfunction.

Inhibition of catalase activity with 3-amino-1,2,4-triazole intensifies bisphenol A (BPA)-induced toxicity in granulosa cells of female albino rats.

Exposure to bisphenol A (BPA), an endocrine disruptor and environmental toxicant, is associated with adverse estrogenic effects in both humans and wildlife species. Because the effects of BPA on the ovary at the cellular level are incompletely understood, the present study was designed to investigate the underlying mechanism of granulosa cell injury following BPA exposure. Eight-week-old female Wistar rats were treated with BPA (25 mg/kg BW/day for 9 days, intraperitonially) with or without pretreatment of the catalase-specific blocker 3-amino-1,2,4-triazole (ATZ; 1 g/kg BW/day for 5 days, intraperitonially). Different oxidative and antioxidant stress parameters, pro-inflammatory cytokines, and hormonal levels were measured. Catalase expression in isolated granulosa cells was analyzed by Western blot. There were noticeable increases in both nitric oxide and lipid peroxidation levels in the granulosa cells of the BPA-treated group with or without pretreatment with ATZ. Compared with the controls, BPA exposure resulted in a significant increase in pro-inflammatory cytokine levels that was further increased following pretreatment with ATZ. Results of the hormonal assays clearly showed a significant decrease in both estrogen and progesterone levels. In contrast, there was a significant increase in both serum follicle-stimulating hormone and luteinizing hormone levels following BPA exposure, with or without ATZ pretreatment. Results of Western blot analysis demonstrated decreased expression of catalase in the BPA-treated group and a further decrease in expression in the group treated with both BPA and ATZ. Our data suggest that catalase plays a role in mediating reproductive damage to granulosa cells exposed to BPA.

A Novel Ligand of Toll-like Receptor 4 From the Sheath of Wuchereria bancrofti Microfilaria Induces Proinflammatory Response in Macrophages.

Background: Lymphatic filariasis, frequently caused from Wuchereria bancrofti infection, is endemic in several parts of the globe and responsible for human health problems and socioeconomic loss to a large extent. Inflammatory consequences originating from host-parasite interaction play a major role in the disease pathology and allied complications. The identity of the key mediator of this process is yet unknown in filarial research. Methods: Microfilarial protein (MfP) was isolated from the sheath of W. bancrofti microfilariae through ultrafiltration, followed by chromatographic separation. Expression of signaling molecules was studied by enzyme-linked immunosorbent assay and immunoblotting. Binding of MfP to Toll-like receptor 4 (TLR4) was determined by co-immunoprecipitation, fluorescein isothiocyanate-probing, and surface plasmon resonance analysis. Results: We found that MfP (approximately 70 kDa) binds to macrophage-TLR4 and triggers nuclear factor kappa beta activation that upregulates secretion of proinflammatory cytokines. Microfilarial protein failed to induce inflammation in either TLRKO macrophage or macrophage treated with TLR4 inhibitor, indicating that MfP acts through TLR4. We have also detected phenotypic transformation of macrophages from anti-inflammatory (M2) to proinflammatory (M1) subtype after incubation with MfP. Conclusions: Microfilarial protein appears to be a new ligand of TLR4 from W. bancrofti. Determination of its functional attributions in the host-parasite relationship, especially immunopathogenesis of filarial infection, may improve our understanding.

Palmitate induced Fetuin-A secretion from pancreatic ß-cells adversely affects its function and elicits inflammation.

Islets of type 2 diabetes patients display inflammation, elevated levels of cytokines and macrophages. The master regulator of inflammation in the islets is free fatty acids (FFA). It has already been reported that FFA and TLR4 stimulation induces pro-inflammatory factors in the islets. In this report we demonstrate that excess lipid triggers Fetuin-A (FetA) secretion from the pancreatic ß-cells. Palmitate treatment to MIN6 cells showed significantly elevated FetA levels in respect to their controls. Fatty acid induces the FetA gene and protein expression in the pancreatic ß-cells via TLR4 and over-expression of NF-κB. In the NF-κB knocked down MIN6 cells palmitate could not trigger FetA release into the incubation medium. These results suggest that NF-κB mediates palmitate stimulated FetA secretion from the pancreatic ß-cells. Blocking the activity of TLR4 by CLI-095 incubation or TLR4 siRNA restored insulin secretion which confirmed the role of TLR4 in FFA-FetA mediated pancreatic ß-cell dysfunction. Palmitate mediated expression of NF-κB enahnced inflammatory response through expression of cytokines such as IL-1ß and IL-6. These results suggest that FFA mediated FetA secretion from pancreatic ß-cells lead to their dysfunction via FFA-TLR4 pathway. FetA thus creates an inflammatory environment in the pancreatic islets that can become a possible cause behind pancreatic ß-cell dysfunction in chronic hyperlipidemic condition.